Acetylene reduction assays (ARAs) and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed to determine the effect of exogenous nitrogen sources on nitrogen fixation activity in three Nodularia spumigena isolates (KAC11, KAC71, and KAC66). It was observed that nitrogen fixation activity decreased in all three isolates in the presence of relatively high levels of ammonium (0.5 mM and 1.0 mM NH4Cl). Concomitantly, real-time RT-PCR analysis showed that expression of nifH, which encodes the dinitrogenase reductase component of the nitrogenase enzyme, decreased in response to ammonium treatment. On the other hand, nitrate (NaNO3) does not seem to affect the nitrogen fixation process. All isolates continuously expressed hetR, encoding the master gene of heterocyst differentiation, and maintained heterocyst frequency along the filaments both in the presence of ammonium and nitrate, even though nitrogen fixation had ceased. Furthermore, expression of ndaF, a marker gene for nodularin production, dropped in the presence of ammonium, indicating a possible correlation between nitrogen fixation and toxin production in these N. spumigena isolates.